Project Summary/Abstract Treponema pallidum (Tp), the causative agent of syphilis, is one of most poorly understood of all bacterial pathogens. That Tp cannot be cultivated continuously in vitro has severely hampered progress towards understanding many of the complexities associated with Tp membrane biology and syphilis pathogenesis. Tp encodes a large number of membrane lipoproteins (LPs) that have widespread importance. Over the past funding interval, we have pursued a structural biology approach for characterizing the Tp LPs by solving their three-dimensional structures and then testing hypotheses about their possible functions. We now have compelling evidence that this unconventional approach represents a successful discovery platform. First, we have described in Tp a periplasmic transport system comprised of two LPs (TP0956/TP0957) that make up a TPR-protein-associated TRAP transport system. This system has many molecular features never before described for a bacterial transport apparatus. TP0956/TP0957 forms a complex that spans precisely the width of the Tp periplasm, and which contains a hydrophobic pore; we shall attempt to determine the ligand carried by this transport complex (Aim 3). We also have identified an essential ABC-type of riboflavin transporter (RfuABCD) in pathogenic spirochetes. This type of riboflavin transport system has never been previously described in bacteria, but it has prompted a re-examination of the potential importance of flavin-containing cofactors and flavoproteins to Tp biology. We also have discovered in Tp a novel FAD pyrophosphatase (TP0796/Ftp), the first such recognition of this type of enzymatic activity in any bacterium; the discovery has further implications for flavin utilization/homeostasis in Tp and, along with our other findings, engenders a new conception that Tp has evolved a flavin-centric lifestyle. Indeed, we have found that Tp encodes a homolog of RnfD (TP0151) that, in other bacteria, requires FMN as a co-factor and typically is a member of an RnfABCDGE operon for redox-driven ion pumps. Tp has been thought NOT to encode such as system, but we also have found a homolog of RnfC (TP0152) (within the gene cluster tp0147-tp0153). These combined findings engender strong supposition that Tp encodes a complete Rnf redox system, something that challenges existing dogma. Establishing that one or more of these Rnf-like proteins interact with each other will provide further evidence of an active Rnf-like redox system in Tp (Aim 2). A better understanding of this flavin-centric lifestyle for Tp may result in valuable new intervention strategies that target either riboflavin uptake or flavin utilization pathways, and could also provide further nutritional information leading to the eventual in vitro cultivation of Tp (Aim 1). Finally, all of our discoveries underscore the power of our structure-to-function approach, and thus we anticipate many more new advancements from discerning the structures of the Tp LPs (Aim 4.).